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  Phosphorylated glyoxalase i and its useUSPTO Application #: 20070059772Title: Phosphorylated glyoxalase i and its use Abstract: The present invention relates to a phosphorylated form of mammalian glyoxalase I. The present invention relates further to the use of phosphorylated mammalian glyoxalase I to modulate MG-modification of proteins (AGE formation) and consequent cell death, especially upon stress such as oxidative stress, or upon TNF treatment. (end of abstract) Agent: Trask Britt - Salt Lake City, UT, US Inventor: Katia Vancompernolle USPTO Applicaton #: 20070059772 - Class: 435007100 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay The Patent Description & Claims data below is from USPTO Patent Application 20070059772. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional of co-pending application Ser. No. 10/630,451, filed Jul. 30, 2003, which is a continuation of co-pending International Patent Application No. PCT/EP02/01118 filed on Jan. 30, 2002 designating the United States of America (International Publication No. WO 02/061065 published in English on Aug. 8, 2002), which claims priority to European Patent Application No. 01200353.9, filed Jan. 31, 2001, the contents of the entirety of each of which are incorporated herein by this reference in their entirety. TECHNICAL FIELD [0002] The present invention relates generally to biotechnology, and, more particularly, to a phosphorylated form of mammalian glyoxalase I. The present invention relates further to the use of phosphorylated mammalian glyoxalase I to modulate methylglyoxal (MG)-modification of proteins and consequent cell death, especially upon stress such as oxidative stress, or upon TNF treatment. BACKGROUND [0003] Tumor Necrosis Factor (TNF) is a pleiotropic cytokine, originally described for its ability to cause hemorrhagic necrosis of certain tumors in vivo (Carswell et al., 1975). In addition to its anti-tumor and anti-malignant cell effects, TNF has been reported to influence mitogenesis, differentiation, and immunoregulation of various cell types. [0004] The activities of TNF are mediated through two cell-surface receptors, namely TNF-R55 (CD120a) and TNF-R75 (CD120b), which are expressed by most cell types. TNF's effects are mediated primarily through TNF-R55. Upon activation of the receptor, adaptor proteins such as TRADD and TRAF are recruited and bind to the intracellular part of the clustered receptor (for review, see Wallach et al., 1999). Those receptor-associated molecules that initiate signaling events are largely specific to the TNF/nerve growth factor receptor family. However, the downstream signaling molecules are not unique to the TNF system, but also mediate effects of other inducers. Downstream signaling molecules in the TNF system identified so far include: caspases, phospholipases, the three mitogen-activated protein (MAP) kinases, and the NF-.kappa.B activation cascade. [0005] TNF-induced cell death in L929 cells is characterized by a necrosis-like phenotype and does not involve DNA fragmentation (reviewed by Fiers et al., 1999). It is independent of caspase activation and cytochrome c release, but is dependent on mitochondria and is accompanied by increased production of reactive oxygen intermediates (ROI) in the mitochondria that are essential to the death process (Goossens et al., 1995; Goossens et al., 1999). The latter was demonstrated by the fact that lipophylic anti-oxidantia, when added three hours after TNF treatment, could not only arrest the ongoing increased ROI production, but could also arrest cell death (Goossens et al., 1995). Furthermore, the mitochondria translocate from a dispersed distribution to a perinuclear cluster (De Vos et al., 2000); functional implications of this mitochondrial translocation remain unclear. [0006] Glyoxalase I, together with glyoxalase II, constitutes the glyoxalase system that is an integral component of the cellular metabolism of .alpha.-ketoaldehydes and is responsible for the detoxification of the latter. The prime physiological substrate of the glyoxalase system is methylglyoxal (MG), which is cytotoxic. The major source of intracellular MG is the glycolysis namely, nonenzymatic and enzymatic elimination of phosphate from dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The glyoxalase system, using glutathione (GSH) as cofactor, catalyzes the conversion of methylglyoxal to D-lactate in two consecutive steps. Glyoxalase I catalyzes the isomerization of the hemithioacetal, produced by the nonenzymatic conjugation of methylglyoxal with glutathione (GSH), to S-D-lactoylglutathione which is then hydrolyzed by glyoxalase II to D-lactate and GSH. D-lactate is then further metabolized to pyruvate by 2-hydroxy-acid dehydrogenase localized in the mitochondria. In addition to its role as a detoxification system, it has been suggested that glyoxalase I, together with its substrate MG, is involved in the regulation of cellular growth (for a review, see Kalapos, 1999), but until now this role has not been found. Increased expression of glyoxalase I occurs in diabetic patients and in some types of tumors such as colon carcinoma (Ranganathan et al., 1993), breast cancer (Rulli et al., 2001), prostate cancer (Davidson et al., 1999). It is also uniquely overexpressed in invasive human ovarian cancer compared to the low malignant potential form of this cancer (Jones et al., 2002). Also, hypoxia can lead to increased expression of glyoxalase I (Principato et al., 1990). Recently, it has been shown that glyoxalase I is involved in resistance of human leukemia cells to anti-tumor agent-induced apoptosis (Sakamoto et al., 2000). DISCLOSURE OF THE INVENTION [0007] While much effort has been directed at the molecular mechanism of the caspase-dependent cell death pathway, relatively little is known about the TNF-induced ROI-dependent cell death pathway. To identify molecules involved in the latter, we performed a comparative study of the phosphoproteins from TNF-treated and control cells by two-dimensional (2-D) gel electrophoresis. It is known that upon activation of the TNF receptor, several kinases/phosphatases are activated (Guy et al., 1992; Guy et al., 1991). However, most of the changes in phosphorylation occur very rapidly (2 to 15 minutes) upon binding of TNF to its receptors and most of them are transient and related to the gene-inductive activities of TNF. [0008] To identify molecules that are involved in the cytotoxic process downstream of the receptor-proximal events, lysates from cells that had been stimulated with TNF for 1.5 hours are studied. Previously, oncoprotein 18 (Op18, stathmin) has been identified as a protein with reproducible and large increases in phosphorylation upon TNF treatment. Op18 is responsible for TNF-induced microtubule stabilization that promotes cell death (Vancompenolle et al., 2000). Unexpectedly, we were able to demonstrate that glyoxalase I is also phosphorylated upon TNF treatment. Phosphorylation of mammalian glyoxalase I has not yet been described, although the sequence does contain several potential phosphorylation sites (Ranganathan et al., 1993). Interestingly, phosphorylation of yeast GLO1 has been observed during the sexual response of S. cerevisiae--specifically, during the arrest of cell division at the G1 phase, which occurs when haploid cells of one sex are exposed to the mating factor of the opposite type of cells (Inoue et al., 1990). However, none of these observations suggest that phosphorylated mammalian glyoxalase I does exist, nor do these observations suggest which potential phosphorylation sites may be used. [0009] It is a first aspect of the invention to provide phosphorylated mammalian glyoxalase. The phosphorylation may be a single or a multiple phosphorylation. An exemplary embodiment is a phosphorylated mammalian glyoxalase I comprising SEQ ID NO:1 of the incorporated herein SEQUENCE LISTING. Preferably, phosphorylated mammalian glyoxalase I essentially consists of SEQ ID NO:1. Even more preferentially, phosphorylated mammalian glyoxalase I consists of SEQ ID NO:1. Preferably, phosphorylation is carried out at position Ser 8 and/or Ser 21 and/or Ser 26 and/or Thr 107. Even more preferably, the phosphorylation is carried out at the PKA phosphorylation sites Ser 45 and/or Thr 98 (numbering as for human glyoxalase, including the N-terminal Met residue). [0010] Another aspect of the invention is the use of a phosphorylated glyoxalase I to modulate MG-modification of proteins. Phosphorylated glyoxalase I may be any glyoxalase I, known to the person skilled in the art, such as a fungal glyoxalase I or a plant glyoxalase I. Preferably, glyoxalase I is a mammalian glyoxalase I. MG-modified proteins or advanced glycation end products (AGEs) are known to be synthesized in response to a number of pathophysiological conditions in vivo, such as cataract formation (Shamsi 2000), vascular complications associated with chronic diabetes (Shinohara et al., 1998), tissue damage after ischemia/reperfusion (Oya et al., 1999) and aging (Corman et al., 1998). The term "AGE," as used here, is used for any MG-modification of a protein, irrespective of the way it is formed. The term "MG-modification of proteins" is considered as being equivalent with the term AGE formation. [0011] Still another aspect of the invention is the use of phosphorylated glyoxalase I, or an inhibitor of the phosphorylation of glyoxalase I, preferably mammalian glyoxalase I to modulated TNF-induced cell death. This inhibitor can be any inhibitor that inhibits the phosphorylation of glyoxalase I. Preferably, the inhibitor is an inhibitor of the PKA activity. [0012] Alternatively, a mutant form of glyoxalase I may be used that affects phosphorylation in it ("phosphorylation mutant"), i.e., it can no longer be phosphorylated at one or more phosphorylation sites and/or it becomes phosphorylated at other sites. On the basis of the knowledge of the phosphorylation sites, such mutants can be easily constructed by the person skilled in the art and include, as a nonlimiting example, glyoxalase I forms where the Ser 45 and/or the Thr 98 have been replaced by another amino acid or any other mutant that affects phosphorylation on these or other sites. Therefore, another aspect of the invention is the use of a phosphorylation mutant of glyoxalase I, preferably mammalian glyoxalase I, to modulate TNF-induced cell death. This modulation can be realized by replacing the endogenous glyoxalase I by the mutant form, or by expressing the mutant glyoxalase I form beside the endogenous glyoxalase I. [0013] A further aspect of the invention is the use of phosphorylated glyoxalase I, or an inhibitor of the phosphorylation of glyoxalase I, or a phosphorylation mutant of glyoxalase I, to modulate stress-induced cell death. Preferably, the stress is oxidative stress. Oxidative stress, followed by ROI induction and AGE formation is known to occur in several organisms, including plants, yeast, fungi and mammalians. An exemplary embodiment is the use of mammalian phosphorylated glyoxalase I to modulate oxidative stress-induced cell death. [0014] Still another aspect of the invention is the use of PKA to phosphorylate glyoxalase I. By modulating the phosphorylation of glyoxalase I, TNF-induced cell death and stress-induced cell death, preferably oxidative stress, can be modulated. BRIEF DESCRIPTION OF THE FIGURES [0015] FIG. 1: phosphorylation of glyoxalase I in control cells (left panel) and after 1.5 hours of TNF treatment (right panel). [0016] FIG. 2: effect of the glyoxalase I inhibitor S-p-bromobenzylglutathione diester on TNF-induced cytotoxicity in L929s cells, in function of the incubation time. TNF is added at a concentration of 1000 units/ml; the inhibitor is added 1 hour 10 minutes prior to TNF at a concentration of 10 .mu.M (GI10) or 20 .mu.M (GI20). The time scale is calculated from the moment of TNF addition. [0017] FIG. 3: western blots, developed with anti-human glyoxalase I polyclonal antibody, of 2-dimensional gels (pH 3-10) from total cell lysates derived from control cells (C), glyoxalase I inhibitor S-p-bromobenzylglutathione diester treated cells (I), TNF-treated cells (TNF) and cells treated with TNF and glyoxalase I inhibitor (TNF+I). [0018] FIG. 4: effect of different concentrations exogeneously added methylglyoxal on TNF-induced cell death. Measurement after 5.5 hours of incubation with TNF and methylglyoxal at a concentration as indicated. [0019] FIG. 5: effect of different concentrations of the AGE formation inhibitor, aminoguanidine, on the TNF-induced cell death. Measurement after 16 hours of incubation with TNF and aminoguanidine at a concentration as indicated. Continue reading... Full patent description for Phosphorylated glyoxalase i and its use Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Phosphorylated glyoxalase i and its use patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Phosphorylated glyoxalase i and its use or other areas of interest. ### Previous Patent Application: Mixture of binding proteins Next Patent Application: Protein o-sulfonation Industry Class: Chemistry: molecular biology and microbiology ### FreshPatents.com Support Thank you for viewing the Phosphorylated glyoxalase i and its use patent info. 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